Journal of Pharmacognosy and Phytochemistry 2020; 9(2): 1614-1620
E-ISSN: 2278-4136
P-ISSN: 2349-8234
www.phytojournal.com
JPP 2020; 9(2): 1614-1620
Received: 01-01-2020
Accepted: 03-02-2020
Ruchi Chauhan
Department of Biotechnology
Dr YS Parmar University of
Horticulture & Forestry, Nauni,
Solan, Himachal Pradesh, India
Poonam Shirkot
Department of Biotechnology
Dr YS Parmar University of
Horticulture & Forestry, Nauni,
Solan, Himachal Pradesh, India
Micropropagation of endangered medicinal plant
Bacopa monnieri (L.) Pennell
Ruchi Chauhan and Poonam Shirkot
Abstract
Bacopa monnieri (L.) Pennell, commonly known as “Brahmi” is an important medicinal herb of the
family Scrophulariaceae. It has main importance as tonic for nervous disorders and mental diseases. It
helps in improving intelligence, memory and learning ability. In the present work, a protocol for
micropropagation of selected genotype of Bacopa monnieri (L.) Pennell has been standardized. Axillary
buds were used as explants for in vitro plant regeneration studies in Bacopa monnieri (L.) Pennell.
Axillary shoot buds showed best in vitro establishment (87.50%) on MS medium supplemented with 0.5
mg/l BAP and 0.5 mg/l Kn. Best in vitro shoot multiplication was obtained on MS medium supplemented
with 1.0 mg/l BAP and 0.5 mg/l Kn. For in vitro rooting, MS medium supplemented with 0.4 mg/l IAA
was found to be the best medium (93.33%). Regenerated plantlets were successfully acclimatized by
using cocopeat followed by a mixture of soil: sand: FYM in ratio 1:1:1.
Keywords: Brahmi, in vitro, BAP, Kn, 2, 4-D
Corresponding Author:
Ruchi Chauhan
Department of Biotechnology
Dr YS Parmar University of
Horticulture & Forestry, Nauni,
Solan, Himachal Pradesh, India
Introduction
The Bacopa genus consists of 20 species living in warm parts of the world, three of which are
represented in India (Sharma, 2003) [19]. Bacopa monnieri (Linn.) Pennell is one of the most
important medicinal plants which belong to Scrophulariaceae family. This herb is commonly
known by various names such as Brahmi, Brahmi-Sak, Kiru-Brahmi, Neerbrahmi, Samrani
and Safed Chamani. Its English name is thyme-left gratiola and water-hyssop (Anonymous,
1996) [2].
It grows sporadically in moist, wet and marshy areas across India. It is found naturally in West
Bengal, Punjab, Haryana and Himachal Pradesh. Himachal Pradesh is situated between
30012040” to 33012040” north latitude and 75047055 "to 7904020" east longitude (Chauhan,
1999) [5] and here this plant is mainly found in moist places of Una, Hamirpur, Kangra,
Bilaspur, Solan, Sirmaur etc. It grows naturally up to a height of 1000 m in marshy areas in the
subtropical region (Sharma, 2003) [19]. The bloom of this plant occurs in the month of July and
continues till December.
The entire herb is used in Ayurvedic, Unani and Siddha systems of indigenous medicine. It is
astringent, bitter, cold and full of vitamin-C. The activity of the plant, Bacopa monnieri, has
been attributed to a complex mixture of triterpenoid saponins, sterols and alkaloids (Rajani,
2008) [23]. Of these saponins, Bacoside-A and Bacoside-B have been found to be the most
important. Other saponins include bacopasaponins-A, B, C, D, F, G and bacopaside I and II.
Other chemical components of the plant are D-mannitol, herpaponin, betulic acid, alkaloids
brahmine and herpestine, flavonoids, and phytosterols (Prabhuji et al., 2005) [15].
In India the plant is used for various types of skin problems - eczema, psoriasis, boils and
ulcers. It is said to stimulate the growth of skin, hair and nails. Indian pennywort is also often
used as an ointment for chronic arthritis. In Pakistan, the herbal medicine, Brahmi-Buti, is
used for the treatment of skin diseases, leprosy, epilepsy, eczema, asthma, hoarseness of voice,
and nervous system diseases (Shakur et al., 1994) [21].
With the release of new memory drugs in the market, the natural population of Bacopa
monnieri is going to be exploited, which must meet the current requirement of 0.1 million
quintals/year of the herb (Sharma et al., 2010) [20]. Thus there is an urgent need to assess
natural populations, to develop protocols for micropropagation, regeneration and agronomic
practices.
In view of the numerous medicinal applications of Bacopa monnieri and its endangered status,
it becomes necessary to develop micropropagation protocols to preserve germplasm and
distribute it for cultivation in new areas. The present work aims to determine the cultivation
conditions for in vitro propagation of this plant. So this work was done with the objective of
standardization of protocol for in vitro mass multiplication of Bacopa monnieri (L.) Pennell.
~ 1614 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
Materials and Methods
Plant Material
The present investigation was carried out in the Department
of Biotechnology of Dr. Y.S. Parmar University of
Horticulture and Forestry, Nauni, Solan. In the present
investigation the plant material used was selected from the
previous study in which Bacopa monnieri (L.) Pennell
genotype of Khaltoo village, Nauni, Solan was found to be
elite genotype on the basis of the leaf length. The plant
material was collected from the selected site and maintained
in the glasshouse of Department of Biotechnology of Dr. Y.S.
Parmar University of Horticulture and Forestry, Nauni, Solan,
for further studies.
Methodology
Preparation of culture media
The explants were cultured on Murashige and Skoog’s (1962)
[14]
medium supplemented with different concentrations of
growth hormones. To reduce the time for weighing individual
ingredients each time, concentrated stock solution of
analytical grade chemicals (macro and micro elements,
vitamins and growth regulators) were prepared and stored at 4
0
C. Plant growth regulators were prepared fresh each time;
auxins were titrated into solution with ethanol, whereas
cytokinins were dissolved in dilute NaOH and then the final
volume is adjusted with double distilled water. In preparing
medium, each stock is added one by one in specified quantity
in double distilled water, after bringing them to room
temperature. After adding meso-inositol (100.0 mg/l), sucrose
(30.0 g/l) and standard amount of growth regulators, the pH of
the medium was adjusted to 5.6-5.8 with the help of 0.1 N
NaOH and/or 0.1 N HCl. Agar- agar (8.0 g/l) was then added
and dissolved by heating the medium. The medium was
poured into flasks/tubes which were then plugged with nonabsorbent cotton wool. Culture vessels containing the medium
were sterilized in autoclave at pressure of 15 lbs per inch2 at
121 0C for 15-20 minutes. After cooling, the media was stored
in dark at 25 0C + 2 0C for about a week to check the
contamination before use.
fluorescent light of average 2500 lux (cool white fluorescent
tubelight 40 W GE). The proliferation was indicated by
unfurling of leaves, elongation of petiole and widening of leaf
lamina. After four weeks the observations were recorded for
percentage of buds proliferated.
In vitro multiplication of shoots
In vitro shoot multiplication was carried out on the MS
medium supplemented with different concentrations of BAP
(0.5-1.0 mg/l) alone and in combination with constant
concentrations of 2, 4-D (0.2 mg/l) and Kn (0.5 mg/l). The
cultures were kept in culture room and after four weeks
observations were recorded for following parameters:
I. Average number of microshoots per shoot
II. Microshoot length (cm)
In vitro rooting of microshoots
Shoots of variable length ranging in height from 3-4 cm were
excised at different stages of subculturing and transferred to
MS medium supplemented with IAA (0.2-0.6 mg/l) and IBA
(0.2-0.6 mg/l) for root induction. The cultures were
transferred to the culture room and after three weeks
observations were recorded for:
I. Per cent rooting based upon number of shoots forming
roots
II. Numbers of roots per microshoot
III. Root length (cm)
Hardening and Acclimatization
The survival and establishment of plantlets was studied after
transplanting the plantlets into different potting media.
I. Cocopeat
II. Sand
III. Soil
IV. Soil + vermicompost (1:1)
V. Soil + FYM (1:1)
VI. Sand + soil + FYM (1:1:1)
Per cent survival of the transferred plants was recorded after
every 2 weeks.
Sizing and surface sterilization of explant
Young shoots were excised from the plant and leaves were
removed carefully. The shoots were cut into small pieces of
size approximately 1 cm with the help of sterilized scalpel
blade so as at least one axillary bud per shoot is present.
These axillary buds were used as explants. The explants i.e.
shoot buds were thoroughly washed with running tap water to
remove the superficial dust. Then they were initially treated
with a mixture of aqueous surfactant and fungicide i.e. 0.2%
(w/v) Teepol and 0.2% (w/v) bavistin respectively for 10-20
min, followed by washing 3-4 times with distilled water.
Henceforth, the manipulations were carried out under aseptic
conditions under laminar air flow hood. The explants were
then surface sterilized using 0.1% HgCl2 for 1-3 minutes;
finally they were washed 4-5 times with autoclaved distilled
water to remove any traces of sterilants.
Statistical analysis
The experiments were conducted in a completely randomized
design (CRD). The data recorded for different parameters was
subjected to analysis of variance (ANOVA) using completely
randomized design [Gomez and Gomez, 1984 [8] (Appendix1)]. Data transformation was carried out as needed to satisfy
ANOVA requirements. Arcsine transformation was
performed on percentage data (derived from count data) lying
in the range of both zero to thirty per cent and seventy to
hundred per cent while square-root transformation was
performed on data consisting of small whole numbers i.e. data
counting in rare events and for percentage data (derived from
count data) lying within the range of 0 to 30 per cent and 70
to 100 per cent, but not both. The data that have been
transformed were expressed in original units for presentation
in the tables.
In vitro establishment of axillary buds
For the establishment of axillary buds, the sterilized explants
were inoculated on to MS medium supplemented with
variable concentrations of BAP (0.25-1.0 mg/l) alone and in
combination with constant concentrations of NAA (0.20 mg/l)
and Kn (0.50 mg/l). The cultures were then placed in the
culture room under the standard conditions of temperature (25
+ 2°C) for 16/8 hrs of day/night break under cool white
Results
Micropropagation of the selected genotype of Bacopa
monnieri (L.) Pennell from Khaltoo village, Nauni, Solan may
lead to mass production of the plants to overcome the
increasing demand and unscientific exploitation which has
given the plant species an endangered status. The information
on various stages of micropropagation of Bacopa monnieri
~ 1615 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
(L.) Pennell and the results obtained during the course of this
investigation are hereunder:
Establishment of shoot buds
For establishment of shoot buds, the sterilized explants were
cultured on MS medium supplemented with variable
concentrations BAP (0.25-1.0 mg/l) alone and in combination
with constant concentrations of NAA (0.2 mg/l) and Kn (0.5
mg/l). A total of 13 treatments were given. Observations on
sprouting of buds were recorded after four weeks and the
results are presented in Table 1.
It has been observed that plant growth regulators had
significant effect on per cent establishment of explants, while
control medium (MS basal medium without growth
regulators) did not show any response of establishment. Bud
initiation was found to be started within 9-10 days by showing
a small newly sprouted bud, which proliferated into shoot
buds with leaves within 20-25 days. A maximum of 87.50%
sprouting of buds was observed with treatment T11 (MS basal
+ 0.5 mg/l BAP + 0.5 mg/l Kn) followed by treatment T9 (MS
basal + 1.0 mg/l BAP + 0.2 mg/l NAA) where percentage of
bud sprouting was found to be 83.33. With treatment T12 (MS
basal + 0.75 mg/l BAP + 0.5 mg/l NAA), T10 (MS basal +
0.25 mg/l BAP + 0.5 mg/l Kn) and T13 (MS basal + 1.0 mg/l
BAP + 0.5 mg/l Kn) sprouting percentage was found to be
79.16, 70.83 and 66.66 respectively. However, minimum per
cent sprouting was observed with treatment T2 (MS basal +
0.25 mg/l BAP) which was found to be 33.33 (Table 1).
Multiplication of in vitro raised shoots
For multiplication, the proliferated in vitro axillary shoots
obtained on establishment medium were transferred to MS
medium supplemented with variable concentrations of BAP
(0.5-1.5 mg/l) alone and with constant concentrations of 2, 4D (0.2 mg/l) and Kn (0.5 mg/l). A total of 10 treatments were
given and observations for average number of shoots per
explant and shoot length were recorded after 4 weeks and the
results are presented in Table 2.
It has been observed that control medium have no effect on
multiplication of microshoots however, maximum average
number of microshoots per explant (7.0) and maximum
average microshoot length (3.00 cm) was obtained with the
treatment T9, supplemented with 1.0 mg/l BAP and 0.5 mg/l
Kn. It was followed by treatment T6 (MS basal + 1.0 mg/l
BAP + 0.2 mg/l 2, 4-D) which resulted in 6.30 microshoots
per explant and length of microshoot was found to be 3.80
cm. With the treatments T10 (MS basal + 1.5 mg/l BAP + 0.5
mg/l Kn), T7 ( MS basal + 1.5 mg/l + 0.2 mg/l 2, 4-D), T5 (
MS basal + 0.5 mg/l BAP + 0.2 mg/l 2, 4-D) and T8 (MS
basal + 0.5 mg/l + 0.5 mg/l Kn) average number of
microshoots per explant were found to be 5.93, 5.86, 5.66 and
5.53 respectively and microshoot length obtained with these
treatments were 2.84 cm, 2.78 cm, 2.60 cm and 2.24 cm
respectively. However, average minimum number of 3.33
microshoots per explant were obtained with treatment T2 (0.5
mg/l BAP) with microshoot length of 1.12 cm (Table 2).
Therefore, treatment T9 was found to be the best treatment for
multiplication of microshoots but for elongation of
microshoots, treatment T6 yielded best results.
Rooting of microshoots
For in vitro root induction microshoots were transferred to
MS basal medium supplemented with different concentrations
of IAA and IBA. A total of five treatments were given and
experiment was carried out to study the effect of different
treatments on per cent in vitro rooting of microshoots, average
root length and average number of roots per shoot (Table 3).
The rooting was initiated after 5 days of incubation and full
rooting was obtained after a period of 30 days. Maximum
rooting per cent of 93.33 was obtained in treatment T6
comprising of 0.4 mg/l IAA. With this treatment maximum
average number of roots per shoot was found to be 6.00 and
their root length was 1.64 cm. This was followed by 86.66 per
cent rooting, average root number of 5.66 and mean root
length of 1.53 cm with treatment T3 comprising of MS
medium supplemented with 0.4 mg/l IBA. With the treatment
T7, T5 and T4 per cent rooting was found to be 80.00, 60.00
and 73.33 respectively and number of roots per shoot
observed was 5.33, 5.00 and 5.13 respectively whereas mean
root length with these treatments were 1.33 cm, 1.20 cm and
1.26 cm respectively. However, minimum rooting was
observed in treatment T2 where per cent rooting was found to
be 66.66, number of roots per shoot were 4.66 and mean root
length observed was 1.13 cm respectively.
Therefore, in vitro rooting in microshoots of Bacopa monnieri
(L.) Pennell was observed on MS medium supplemented with
4.0 mg/l IAA. The fully developed plantlets were obtained
after one month.
Hardening of in vitro raised plantlets of Bacopa monnieri
(L.) Pennell
Fully developed in vitro raised plantlets obtained after the
previous stage of in vitro rooting needed to be hardened and
acclimatized. Hardening of the plantlets was carried out to
acclimatize in vitro raised plantlets to in vivo conditions. After
four weeks of incubation on rooting medium, plantlets of size
about 3 cm with well developed roots were removed from the
culture medium very carefully and the roots were washed
gently under running tap water to remove the sticking
medium. The plantlets were then dipped in 0.2% bavistin for
1-5 min followed by transferring to the plastic cups
containing different hardening mixtures. Different hardening
mixtures investigated consisted of cocopeat, sand and soil and
their combinations. This experiment was carried out to study
the suitability of these hardening mixtures on the per cent
survival of the regenerated plantlets and results obtained have
been presented in Table 4. Perusal of the data presented in the
Table 4 shows a comparison of survival of in vitro raised
plantlets in the sterilized substrates after six weeks of transfer
from in vitro conditions to plastic cups kept in the culture
room for 10-15 days.
It has been observed that per cent survival of the plantlets in
the cocopeat is much higher as compared to the survival
percentage of plantlets in other substrates. Total of 80 per cent
plantlets survived after 6 weeks when hardened in cocopeat.
Sand and soil resulted in 68 per cent and 42 per cent survival
of the plantlets after 6 weeks of their transfer. When sand and
soil were used as a hardened mixture in ratio 1:1, per cent
survival observed was 45 whereas 48 per cent of plantlets
survived in a mixture of cocopeat and sand in ratio 1:1, after 6
weeks of transfer to this mixture.
Therefore, cocopeat was found to be most suitable hardening
mixture for the acclimatization of in vitro raised plantlets and
was selected as the hardening mixture for the further
acclimatization of plantlets. Hardened plantlets were
transferred to small pots containing the sterilized mixture of
soil: sand: FYM (1:1:1) and these were covered with glass
jars and were transferred to glass house.
~ 1616 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
Acclimatization of in vitro raised hardened plantlets
The hardened plantlets which were transferred to small pots
containing soil: sand: FYM in 1:1:1 ratio was kept covered
with glass jars for 1 week and then the jars were removed and
the pots were kept in the glass house. The growth rate and
survival of the plantlets were observed for 2, 4, 6 and 8 weeks
and the observations have been presented in Table 5.
Perusal of data presented in table 11 reveals that per cent
survival of in vitro raised hardened plantlets after 2nd week of
transplantation was found to be 91.67% with plant height of
2.96 cm and average number of leaves per plant were 19.50.
After 4th week the survival per cent decreased and reached to
83.33% but plant height was increased to 4.22 cm and number
of leaves per plant also increased to 25.50. After 6th week per
cent survival remained to 75% but plant height increased
considerably and reached to 5.18 cm. Number of leaves has
also increased to 31.00. Per cent survival decreased with
passage of time and remained only 66.67% after 8th week of
transfer to pots, with mean plant height of 6.08 cm. However,
there is a remarkable increase in average number of leaves per
plant. After 8th week average numbers of leaves per plant
observed were 39.00.
Discussion
The first step towards in vitro establishment of cultures is an
appropriate choice of explant. The available literature has
shown that in case of medicinal plants, nodal and internodal
segments have been successfully used as explants (Razdan,
1993) [16]. It has been revealed that micropropagation of
Bacopa monnieri could be carried out using axillary buds and
internodal segments for producing true to type plants.
After excision of the explants, the most critical factor for
successful establishment of cultures is the surface sterilization
of the explants, since contaminants usually reduce the
establishment rate of in vitro cultures. Excised shoot buds and
leaves of Bacopa monnieri were surface sterilized using
different sterilization treatments of a fungicide bavistin
followed by HgCl2. The maximum percent of uncontaminated
cultures was achieved by using 0.2% bavistin for 15 min
followed by 0.1% HgCl2 for 2 min and finally washing with
autoclaved distilled water 4-5 times. This method of
sterilization has been found to be working satisfactorily
provided enough care taken to remove residues of sterilants
by proper washing with sterile water that otherwise have
inhibitory effect on further growth of the explants whereas
Sharma et al. (2010) [20] reported use of 1-2% cetavelon
detergent solution for 10 min followed by 5 min treatment
with 0.1% HgCl2. Explants were surface sterilized with 50%
ethanol followed by a 3 min treatment with 0.01% was
reported by (Binita et al., 2005) [3]. Some authors have
reported 0.1% (w/v) HgCl2 along with other surface sterilants
for sterilization of explants of various medicinal plant species
such as Gloriosa superb (Hassan and Roy, 2005) [12],
Plumbago zeylanica (Chinnamadasamy et al., 2010) [6],
Phyllanthus amarus (Ghanti et al., 2004) [7], Clitoria ternatea
(Rout, 2004) [17], Nyctanthes arbortristis (Rout et al., 2007)
[18]
, Ricinus communis (Alam et al., 2010) [1], Portulaca
grandiflora (Jain and Bashir, 2010) [13], Solanum nigrum
(Sundari et al.,2010) [24] and Boesenbergia rotunda (Yusuf et
al., 2011) [27]. Surface sterilization with HgCl2 in case of
Bacopa monnieri explants have also been reported by Sharma
et al., 2010 [20].
In the present investigation, a protocol for establishment of
axillary shoot buds and in vitro shoot multiplication has been
developed using MS basal medium supplemented with
different concentrations of growth regulators - BAP, Kn and
2, 4- D. Treatment T11 comprising of 0.50 mg/l BAP and Kn
was found to be the best performing medium for
establishment of axillary shoot buds giving a maximum value
of 87.50 per cent of proliferated buds after seven days of
culturing (Table 1). Earlier studies have shown that BAP
alone could be used for in vitro shoot proliferation e.g.
Sharma et al., 2010 [20] reported the use of BAP alone for the
axillary bud break and shoot proliferation, they found out that
0.2 mg/l concentration of BAP results in 13-15 folds shoot
proliferation. These results are in line with those workers,
indicating the efficiency of BAP for shoot culture initiation
and multiplication in Bacopa monnieri, reported by (Tiwari et
al., 2000; Srivastava, 1999) [26, 23].
Development of rootlets leads to full plantlets and in vitro
root induction is carried using growth regulators such as
auxins which include IBA, IAA and NAA. In the present
study 0.4 mg/l IAA produced maximum root formation of
93.33 per cent, whereas Sharma et al. (2010) [20] reported use
of IBA in MS medium for best rooting. Different authors
reported usage of IAA either alone or in combination with
IBA for successful rooting in several medicinal plants such as
Plumbago zeylanica (Chinnamadasamy et al., 2010) [6],
Nyctanthes arbortristis (Rout et al., 2007) [18], Bauhinia
cheilantha (Gutierrez et al., 2011) [10] and Solanum nigrum
(Sundari et al., 2010) [24].
Most of the mortality of micropropagated plantlets occurs at
the acclimatization stage. A substantial number of
micropropagated plantlets do not survive the transfer from in
vitro conditions to green house or field environment.
Therefore, transferring of in vitro regenerated plantlets to soil
remains a crucial step in micropropagation (Grout, 1975;
Sutter and Langhans, 1982; Short et al., 1987) [9, 25, 22] due to
reduced amounts of epicuticular wax and reduced vascular
tissue developed. But such features pose no problem in in
vitro conditions when microplants are surrounded by high
humidity. In the present study the plantlets with well
developed shoots and roots were able to survive when
hardening was carried out in pots containing autoclaved
mixture of soil: sand: FYM in the ratio of 1:1:1 with decrease
in survival from 91.67% to 66.67% within a time period of 28 weeks. High humidity helped in enhancing the survival of
plants by covering them with glass jars which were uncovered
after about 10 days of transfer to soil and acclimatization of
plantlets on the similar mixture has been reported by Hamirah
et al. (2010) [11] in Zingiber montanum. However, in another
report given by Chan et al. (2009) [4], the in vitro regenerated
plantlets of Gynura procumbens were acclimatized in organic
soil: sand mixture (1:1).
Thus in the present study a protocol has been developed
successfully for micropropagation of Bacopa monnieri (L.)
Pennell genotype of Khaltoo village Nauni, which can be
further used for mass production of this genotype of this
threatened plant species.
~ 1617 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
Fig 1: A&B. In vitro establishment of cultures from shoot buds of Bacopa monnieri (Linn.) Pennell. C&D. In vitro multiplied shoots of Bacopa
monnieri (Linn.) Pennell E. Rooting of in vitro raised shoots F. Hardening of Bacopa monnieri plantlets in mixture of Soil: FYM: Sand
Table 3: Effect of different concentrations of IBA and IAA on in
vitro root induction in microshoots of Bacopa monnieri (L.) Pennell.
Table 1: Effect of various concentrations and combinations of
growth regulators on the establishment of shoot buds of Bacopa
monnieri (L.) Pennell after four weeks of culturing.
Treatment
(T)
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Plant growth regulators
BAP
NAA
Kn
(mg/l)
(mg/l)
(mg/l)
0.25
0.50
0.75
1.00
0.25
0.20
0.50
0.20
0.75
0.20
1.00
0.20
0.25
0.50
0.50
0.50
0.75
0.50
1.00
0.50
CD0.05
SE+
Treatment
Percentage of buds
proliferated
1
2
3
4
5
6
7
CD 0.05
SE+
0.000(0.000)*
33.33 (35.20)
41.66 (40.20)
45.83 (42.61)
54.16 (47.39)
50.00 (45.00)
58.33 (49.80)
62.50 (52.24)
83.33 (65.90)
70.83 (57.32)
87.50 (69.30)
79.16 (62.84)
66.66 (54.73)
0.79114
0.38488
Table 4: Effect of hardening mixtures on acclimatization of in vitro raised
plantlets of Bacopa monnieri (Linn.) Pennell after six weeks of incubation.
Table 2: Effect of various concentrations and combinations of
growth regulators on the multiplication of microshoots of Bacopa
monnieri (L.) Pennell after four weeks of culturing.
1
2
3
4
5
6
7
8
9
10
Root length
(cm)
0.000
1.130
1.530
1.260
1.200
1.640
1.330
0.17171
0.080060
*Values expressed in parentheses are arc sine transformation of
percentage
*Values expressed in parentheses are the arc sine transformation of
percentage.
Treatment
(T)
Phytohormone
No. of roots
Percent rooting
per shoot
IBA
IAA
0.00(0.00)*
0.000
0.2
66.66(54.74)
4.660
0.4
86.66(68.60)
5.660
0.6
73.33(58.91)
5.130
0.2
60.00(50.77)
5.000
0.4
93.33(75.07)
6.000
0.6
80.00(63.44)
5.330
1.4654
0.62625
0.68323
0.29199
Plant growth regulators
Average no. of
Microshoot
microshoots
BAP
2,4-D
length (cm)
Kn (mg/l)
per explant
(mg/l)
(mg/l)
0.00
0.00
0.50
3.33
1.06
1.00
4.13
1.11
1.50
4.66
1.12
0.50
0.20
5.66
2.60
1.00
0.20
6.30
3.80
1.50
0.20
5.86
2.78
0.50
0.50
5.53
2.24
1.00
0.50
7.00
3.00
1.50
0.50
5.93
2.84
CD 0.05
0.16282
0.15867
S.E+
0.078056
0.07552
Substrate
Per cent survival
Cocopeat
80
Sand
68
Soil
42
Sand + Soil (1:1)
45
Cocopeat + sand (1:1)
48
CD0.05
SE +
*Values expressed in parentheses are arc sine transformation of
percentage
Table 5: Effect of hardening at weekly interval on the growth and
development of Bacopa monnieri (Linn.) Pennell
Treatments
Per cent
Plant height No. of leaves per
(weekly)
survival
(cm)
plant
2th
91.67 (73.26)*
2.96
19.50
4th
83.33 (65.91)
4.22
25.50
6th
75.00 (60.00)
5.18
31.00
8th
66.67 (54.74)
6.08
39.00
CD 0.05
1.7186
1.8499
1.9789
SE +
0.74526
0.80223
0.85816
*Values expressed in parentheses are arc sine transformation of
percentage
~ 1618 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
Table 8 E: Effect of hardening at weekly interval on growth and
development of Bacopa monnieri (Linn.) Pennell.
Anova Tables
Table 1 A: Effect of various combinations of growth regulators on
the establishment of shoot buds of Bacopa monnieri (Linn.) Pennell.
Source of variation Degree of freedom
Treatment
Error
Total
12
26
38
Treatment
Error
Total
SS
MSS
F
Per cent survival
11140.0 928.30 4177.71
5.7773 0.2222
11145.0
Table 2 B: Effect of various combinations of growth regulators on
multiplication of microshoots of Bacopa monnieri (Linn.) Pennell.
Source of
variation
Degree of
freedom
Treatment
Error
Total
9
20
29
SS
MSS
F
Average number of shoots per
explant
15.931
1.7701
193.69
0.18278
0.0091391
16.114
Table 3 C: Effect of various combinations of growth regulators on
multiplication of microshoots of Bacopa monnieri (Linn.) Pennell.
Source of variation
Degree of
freedom
Treatment
Error
Total
9
20
29
SS
MSS
F
Shoot length
37.174 4.1305 11.04
7.4804 0.37402
44.655
Table 4 D: Effect of different concentrations of IAA and IBA on in
vitro root induction in microshoots of Bacopa monnieri (Linn.)
Pennell.
Source of variation
Degree of
freedom
Treatment
Error
Total
6
14
20
SS
MSS
F
Per cent rooting
11074 1845.7 2635.93
9.8028 0.70020
11084
Table 5 E: Effect of different concentrations of IAA and IBA on in
vitro root induction in microshoots of Bacopa monnieri (Linn.)
Pennell.
Source of variation
Degree of
freedom
Treatment
Error
Total
6
14
20
SS
MSS
F
No. of roots per shoot
75.587 12.598 98.51
1.7904 0.12789
77.378
Table 6 F: Effect of hardening at weekly interval on growth and
development of Bacopa monnieri (Linn.) Pennell.
Source of variation
Degree of
freedom
Treatment
Error
Total
3
8
11
SS
MSS
F
Per cent survival
569.78 189.93 227.97
6.6650 0.83312
576.44
Table 7 G: Effect of hardening at weekly interval on growth and
development of Bacopa monnieri (Linn.) Pennell.
Source of variation
Degree of freedom
Treatment
Error
Total
3
8
11
SS
MSS F
Plant height (cm)
16.081 5.3604 5.55
7.7228 0.9653
23.804
Source of variation
Degree of
freedom
3
8
11
SS
MSS
F
No. of leaves per plant
618.75 206.25 186.71
8.8372 1.1046
627.59
References
1. Alam Iftekhar, Sharmin Shamima Akhtar, Mondal
Sanjoy Chandra, Alam Md. Jahangir, Khalekuzzaman
Muhammad, Anisuzzaman M et al. In vitro
micropropagation through cotyledonary node culture of
castor bean (Ricinus communis L.). Australian Journal of
Crop Science. 2010; 4:81-84
2. Anonymous. Wealth of India. Council of Scientific and
Industrial Research, New Delhi. 1996, 143
3. Binita B Chaplot, Ashok M Dave, Yogesh T Jasrai.
Bacopa monnieri (L.) Pennell: A Rapid, Efficient and
Cost Effective Micropropagation. Plant Tissue Culture
and Biotechnology. 2005; 15(2):167-175
4. Chan Lai Keng, Lim Su Yee. Micropropagation of
Gynura procumbens (Lour.) Merr. an important
medicinal plant. Journal of medicinal plant research.
2009; 3(3):105-111
5. Chauhan NS. Medicinal and aromatic plants of Himachal
Pradesh. Indus Publishing Company, New Delhi. 1999,
234-237
6. Chinnamadasamy Kalidass, Arjunan Daniel, Ramasamy
Mohan Veerabahu. Rapid Micropropagation of
Plumbago zeylanica L. – an important medicinal plant.
Journal of American Science. 2010; 6(10):1027-1031
7. Ghanti Kiran S, Govindaraju B, Venugopal RB, Rao S
Ramgopal, Kaviraj CP, Jabeen FTZ et al. High frequency
shoot regeneration from Phyllanthus amarus Schum. &
Thonn. Indian Journal of Biotechnology. 2004; 3:103107
8. Gomez KA, Gomez AA. Statistical procedures for
agricultural research (2 ed.). John wiley and sons, New
York, 1984, 680.
9. Grout BW. Wax development on leaf surface of Brassica
oleracea var. Curwang regenerated from meristem
culture. Plant Science Letters. 1975; 5:401-405
10. Gutierrez IEM, Nepomuceno CF, Ledo CAS, Santana
JRF. Micropropagation and acclimatization of Bauhinia
cheilantha (an important medicinal plant). African
Journal of Biotechnology. 2011; 10(8):1353-1358
11. Hamirah MN, Sani HB, Boyce PC, Sim SL.
Micropropagation of red ginger (Zingiber montanum
Koenig), a medicinal plant. AsPac Journal of Molecular
Biology and Biotechnology. 2010; 18(1):127-130
12. Hassan AKM, Sayeed, Roy Shyamal K Roy.
Micropropagation of Gloriosa superb L. through high
frequency shoot proliferation. Plant Tissue Culture. 2005;
15(1):67-74
13. Jain Ashok K, Bashir Mudasir. In vitro propagation of a
medicinal plant Portulaca grandiflora. Hook. World
Journal of Agricultural Sciences. 2010; 6(3):327-330
14. Murashige T, Skoog F. A revised medium for rapid
growth of bioassay with tobacco tissue cultures.
Physiologia Plantarum. 1962; 15:473-497
15. Prabhuji SK, Rao GP, Patil SK. Recent advances in
medicinal plants research. Satish Serial Publication, New
Delhi. 2005, 16-20
~ 1619 ~
Journal of Pharmacognosy and Phytochemistry
http://www.phytojournal.com
16. Razdan MK. An introduction to plant tissue culture.
Andover, Hampshire: Intercept, 1993.
17. Rout Gyana Ranjan. Effect of cytokinins and auxins on
micropropagation of Clitoria ternatea L. Biological
Letters. 2004; 41(1):21-26
18. Rout GR, Mahato A, Senapati SK. In vitro clonal
propagation of Nyctanthes arbortristis Linn. -a medicinal
tree. Horticultural Science. 2007; 34(2):84-89
19. Sharma R. Medicinal plants of India. Daya Publishing
House, New Delhi. 2003, 30-31
20. Sharma Sudhir, Kamal Barkha, Rathi Neelima, Chauhan
Sudhir, Jadon Vikas, Vats Neha et al. In vitro rapid and
mass multiplication of highly valuable medicinal plant
Bacopa monnieri (L.) Wettst. African Journal of
Biotechnology. 2010; 9(49):8318-8322
21. Shakoor A, Akram M, Asharaf CM, Siddiqui MR.
Pharmagonistic study and chemical/pharmacological
evaluation of Brahmi-buti. Hamdard Medicus. 1994;
37:92-109
22. Short KC, Warburton J, Roberts AV. In vitro hardening
of cultured cauliflower and chrysanthemum plantlets to
humidity. Acta Horticulturae. 1987; 212:309-344
23. Shrivastava N, Rajani M. Multiple shoot regeneration and
tissue culture studies on Bacopa monnieri (L.) Pennell.
Plant Cell Reports. 1999; 18:919-923
24. Sundari MS, Benniamin A, Manickam VS.
Micropropagation and in vitro flowering in Solanum
nigrum Linn. A medicinal plant. International Journal of
Biological Technology. 2010; 1(1):29-32
25. Sutter E, Langhans RW. Formation of epicuticular wax
and its effect on water loss in cabbage plants regenerated
from shoot tip culture. Canadian Journal of Botany. 1982;
62:2896-2902
26. Tiwari V, Tiwari KN, Singh BD. Suitability of Liquid
cultures for in vitro multiplication of Bacopa monniera
Linn. Wettst. Phytomorphology 2000; 50(3, 4):337-34
27. Yusuf Nor Azma, Annuar MM Suffian, Khalid
Norzulaani. Rapid micropropagation of Boesenbergia
rotunda (L.) Mansf. Kulturpfl. (a valuable medicinal
plant) from shoot bud explants. African Journal of
Biotechnology. 2011; 10(7):1194-1199.
~ 1620 ~